Purification of acidic proteins using ceramic hydroxyapatite chromatography
a technology of hydroxyapatite and acidic proteins, which is applied in the direction of antibody medical ingredients, pharmaceutical active ingredients, separation processes, etc., can solve the problems of adversely affecting product safety, reducing product yield, and reducing product efficacy
Patent Information
- Authority / Receiving Office
- US · United States
- Patent Type
- Patents(United States)
- Current Assignee / Owner
- Publication Date
- 2011-11-15
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Abstract
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. §119(e) to U.S. provisional application No. 61 / 110,468, filed Oct. 31, 2008, which is hereby incorporated by reference in its entirety.FIELD OF THE INVENTION
[0002] The present invention describes a method of removing partially active and / or inactive product-derived species, high molecular weight aggregates, and other impurities from acidic proteins, e.g., Ig-fusion proteins, using ceramic hydroxyapatite chromatography. Under the specific operating binding and elution conditions provided in this invention, acidic protein product, e.g., acidic Ig-fusion protein product, can be separated from the product-derived and process-derived impurities with high resin binding capacity and good product yield.BACKGROUND OF THE INVENTION
[0003] It is desirable to identify useful methods of purifying proteins that do not destroy, or significantly reduce, the biological activity of the protein. Contaminants ...
Examples
example 1
[0062]An acidic immunoglobulin fusion protein, ActRIIB-Fc, was expressed in CHO cells and purified from the harvest media by rProtein A chromatography (GE-Healthcare, Piscataway, N.J.). The protein elution from rProtein A chromatography, which contained ActRIIB-Fc protein, also contained a significant level of inactive and partially active species and HMWA (see “Load” row in Table 2). In order to remove inactive and partially active ActRIIB-Fc species and HMWA, the elution from the Protein A column was subjected to cHA chromatography.
[0063]The cHA column (BioRad Laboratories, Hercules, Calif.) was first equilibrated with Equilibration Buffer 1, containing 0.3 M sodium phosphate, 1.0 M NaCl, pH 6.8, followed by Equilibration Buffer 2, containing 10 mM HEPES, pH 7.2 (Table 1). Equilibration Buffer 1 is a buffer with high concentration of phosphate and the Equilibration Buffer 2 is used to wash out the phosphate. The column was subsequently equilibrated with calcium chloride solution a...
example 2
[0069]cHA chromatography was found to be superior for HMWA removal during purification of another acidic Ig fusion protein comprising IL21 fused to the Fc domain of Ig (sIL21r-Fc). See Ettinger et al., Ann Rheum Dis 2008; 67 (Suppl III):iii83-iii86 (for a description of the sIL21r-Fc fusion protein). One goal for this process step was to remove HMWA in order to improve the subsequent chromatographic step's capacity for HMWA and HCP. The column was equilibrated with a calcium chloride solution at neutral pH and low ionic strength. The rProtein A eluate pool was not spiked with calcium chloride for this process due to observed product instability upon exposure to calcium chloride, but rather, the rProtein A eluate pool was loaded directly onto the cHA column at 20-30 g / L resin load challenge. Under these buffer conditions, the product was bound to the cHA resin, with some species of HMWA flowing through the column. The column was then washed with a neutral pH and low ionic strength bu...